Liposomal delivery system for biologically active agents

ABSTRACT

The present invention is directed to a liposomal preparation which is based on specific lipid components. The liposomal compounds are also combined with a biologically active agent, forming liposomal compounds. These compounds are useful in drug delivery, where specific therapeutic compounds are provided in the liposomes. The specific lipid components of the present invention provide a highly efficient and stable delivery system for nucleic acids. Consequently, one embodiment of the invention provide the liposomal preparations which are suitable for use in gene therapy.

This application is a 371 of PCT/US95/09867 filed Aug. 4, 1995 and aC-I-P of Ser. No. 08/286,730, filed Aug. 5, 1994.

FIELD OF THE INVENTION

The present invention is directed to a liposomal preparation which isbased on a composition of specific lipids which form liposomes. It isalso an object of the present invention to provide a method forpreparing a liposomal composition carrying a biologically active agentwhich is simple and very efficient. The liposomal delivery system of thepresent invention is used as a highly efficient transfer therapy method.

BACKGROUND OF THE INVENTION

Lipidic particles have been shown to be efficient vehicles for many invitro and in vivo applications. Lipidic particles complexed with DNAhave been used in vitro (Felgner P. L., et al. Proc. Natl. Acad. Sci.USA 84, 7413-7417 (1987); Gao X. et al. Biochem. Biophys. Res. Commun.179, 280-285 (1991)) and in vivo (Nabel E. G., et al. Science 249,1285-1288 (1990); Wang C. et al. Proc. Natl. Acad. Sci. USA 84,7851-7855 (1987); Zhu N., et al. Science 261, 209-211 (1993); SorianoP., et al. Proc. Natl. Acad. Sci. USA 80, 7128-7131 (1983)) for theexpression of a given gene through the use of plasmid vectors. Formationof complexes of DNA with cationic lipidic particles has recently beenthe focus of research of many laboratories. In particular, lipofectin™(Gibco BRL, Gaithersburg, Md.) has been successfully used for thetransfection of various cell lines in vitro (Felgner P. L., et al. Proc.Natl. Acad. Sci. USA 84, 7413-7417 (1987)) and for systemic geneexpression after intravenous delivery into adult mice (Zhu N., et al.Science 261, 209-211 (1993)).

Lipidic particles may be complexed with virtually any biologicalmaterial. These particles may be complexed with proteins, therapeuticagents, chemotherapeutic agents, and nucleic acids and provide a usefuldelivery system for these agents. One such drug delivery system, genetherapy, is one such area which has produced promising results. In thisarea two different strategies have emerged: Gene therapy andoligonucleotide-based therapeutics. To be successful these twoapproaches must be mediated by an efficient "in vivo" transfer of thenucleic acid material to the target cells and there is a need to providean efficient and safe delivery system of nucleic materials.

Gene therapy may involve the transfer of normal, functional geneticmaterial into cells to correct an abnormality due to a defective ordeficient gene product. Typically, the genetic material to betransferred should at least contain the gene to be transferred togetherwith a promoter to control the expression of the new gene.

Viral agents have been demonstrated to be highly efficient vectors forthe transfection of somatic cells. Retroviruses in particular havereceived a great deal of attention because they not only enter cellsefficiently, but also provide a mechanism for stable integration intothe host genome through the provirus. However, clinical use ofretroviral vectors is hampered by safety issues. A first concern is thepossibility of generating an infectious wild type virus following arecombination event. A second concern is the consequences of the randomintegration of the viral sequence into the genome of the target cellwhich may lead to tumorigenic event. In addition, as retroviruses wouldonly complete their life cycle in dividing cells, a retroviral vectorwould be inefficient in targeting cells which are not dividing. DNAviruses such as adenoviruses are potential gene carriers but thisstrategy is limited in the size of the foreign DNA adenoviruses cancarry and because of the restricted host range. However, the advantageof adenoviruses over retroviral vectors is their ability to infectpost-mitotic cells.

Synthetic gene-transfer vectors have been subject to intenseinvestigation since this strategy appears to be clinically safe.Potential methods of gene delivery that could be employed includeDNA/protein complexes (Cristiano R. J., et al. Proc. Natl. Acad. Sci.USA 90, 2122-2126 (1993)) or lipidic particles (Nabel E. G., et al.Science 249, 1285-1288 (1990); Felgner P. L., et al. Proc. Natl. Acad.Sci. USA 84, 7413-7417 (1987); Wang C. et al. Proc. Nat. Acad. Sci. USA84, 7851-7855 (1987); Gao X. et al. Biochem. Biophys. Res. Commun. 179,280-285 (1991); Zhu N., et al. Science 261, 209-211 (1993); Soriano P.,et al. Proc. Natl. Acad. Sci. USA 80, 7128-7131 (1983)). The geneticmaterial to be delivered to target cells by these methods are plasmids.Plasmids are autonomous self-replicating extra chromosomal circular DNA.They can be modified to contain a promoter and the gene coding for theprotein of interest. Such plasmids can be expressed in the nucleus oftransfected cells in a transient manner. In rare events, the plasmidsmay be integrated or partly integrated in the cell host genome and mighttherefore be stably expressed. Episomal plasmid vectors are plasmidsable to replicate in the nucleus of the transfected cells and maytherefore be expressed in a total growing cell population. Plasmids havea promising potential considering the fact that they may be applied incombination with a synthetic vector as carrier and that gene therapy bythis means may be safe, durable, and used as drug-like therapy.

Plasmid preparation is simple, quick, safe, and inexpensive representingimportant advantages over retroviral vector strategy. The successful useof this genetic tool for "in vivo" approaches to gene therapy will relyon the development of an efficient cell delivery system.

Retroviral vectors have been shown to be very efficient for genetherapy. However, their use for in vitro human gene therapy has severallimitations. Retroviral vectors may, by insertional mutagenesis lead toactivation of oncogenes and increase the frequency of malignanttransformation. They will not transfect non dividing cells, and theirstability and titer are adversely affected by large gene insert.Adenoviral vectors which give rise to transient expression are currentlylimited by a demonstrated toxicity in vivo. Presently,replication-compromised herpes simplex virus vectors have toxic effectson the cells they infect, thus limiting their use for human trials.These obstacles have led several laboratories to develop physical meansof gene transfer such as the pneumatic DNA gun (Yang et al. 1990 Proc.Natl. Acad. Sci. USA 9568-72), direct DNA injection (Wolff et al. 1990Science 247:1465-68), or liposome delivery vector (Fergner et al. Proc.Natl. Acad. Sci. USA 1987 84:7413-17).

The fact that viral vectors have limitations such as propensity forrecombination, low titer, and induction of host immunity has initiatedresearch into non-viral vectors. The delivery of plasmid DNA viasynthetic carriers to cells "in vivo" by direct i.v. administration isappealing because of its simplicity and potential to reach a far greaternumber of cells than by an "ex vivo" approach. Although the efficiencyof "in vivo" transfection of DNA plasmids is limited when compared todelivery by viral vectors, recent advances, especially in lipidicparticle delivery, have demonstrated that non-viral gene transfer offersexciting potential, including its use in a clinical setting. More recentattempts to deliver gene or antisense oligonucleotides has provided anew impetus to lipid particle technology (Leonetti, J. P., et al. (1990)Proc. Natl. Acad. Sci. USA 87, 2448-2451; Burch, R. M. et al. (1991) J.Clin. Invest. 88, 1190-1196; Thierry, A. R. et al. (1992) Nucleic AcidsRes. 20, 5691-5698; Smith, J. G., et al. (1993) Biochim. Biophys. Acta1154, 327-340). One such approach is based on the formation of complexesof DNA with cationic lipidic particles. A few therapeutic clinical trialprotocols using local administration of these complexes are ongoing butdata on systemic administration is still poorly documented (Wang C. etal. (1987) Proc. Natl. Acad. Sci. USA 84, 7851-7855; Zhu, N. et al.(1993) Science 261, 209-211).

Several lipids have been used in attempts to prepare liposome-likeparticles. One such lipid mixture is Lipofectin™ which is formed withthe cationic lipid DOTMA,N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-ammonium chloride, and DOPE,dioleylphosphatidyl ethanolamine at a 1:1 molar ratio. The lipidicparticles prepared with this formulation spontaneously interact with DNAthrough the electrostatic interaction of the negative charges of thenucleic acids and the positive charges at the surface of the cationiclipidic particles. This DNA/liposome-like complex fuses with tissueculture cells and facilitates the delivery of functional DNA into thecells (Felgner P. L., et al. Proc. Natl. Acad. Sci. USA 84, 7413-7417(1987)). New cationic lipid particles have been developed:Lipofectamine™ (Gibco BRL), composed of DOSPA,2,3-dioleyloxy-N[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoracetate and DOPE at a 1:1 molar ratio. Lipofectace™ (Gibco BRL)composed of DDAB, dimethyidioctadecylammonium chloride and DOPE at a 1:1molar ratio. DOTAP™ (Boehringer Mannheim, Ind.) is 1-2-dioleoyloxy-3(trimethyl ammonia) propane.

Behr et al. (Proc. Natl. Acad. Sci. USA 86, 6982-6986 (1989); BarthelF., et al. DNA Cell Biol. 12, 6, 553-560 (1993)) have recently reportedthe use of a lipopolyamine (DOGS,Spermine-5-carboxy-glycinediotade-cylamide) to transfer DNA to culturedcells. Lipopolyamines are synthesized from a natural polyamine sperminechemically linked to a lipid. For example, DOGS is made from spermineand dioctadecylamidoglycine (Behr J. P., et al. Proc. Natl. Acad. Sci.USA 86, 6982-6986 (1989)). DOGS spontaneously condense DNA on a cationiclipid layer and result in the formation of nucleolipidic particles. Thislipospermine-coated DNA shows high transfection efficiency (Barthel F.,et al. DNA Cell Biol. 12, 6, 553-560 (1993)).

However, the above-described lipid compositions fail to produceliposomes. Rather, these investigations synthesized lipid particles,which are clusters of lipid molecules which have not formed at least alipid bilayer membrane and therefore also lack an aqueous internalspace. Since these particles are mere clusters of lipid molecules, theparticles lack the ability to act as storage units for biologicallyactive agents contained therein.

Therefore it is an object of the present invention to provide aliposomal composition capable of carrying internally biologically activeagents.

It is an other object of the present invention to provide an efficient,stable and safe liposome-based delivery system for biologically activematerials.

Yet another object of the present invention to provide a novel liposomalcomposition comprising a cationic lipopolyamine and a neutral lipid.

It is yet another object of the present invention to provide a method oftransferring biologically active agents into cells and patients usingthe instant liposomal delivery system.

It is a further object of the present invention to provide a method ofpreparing liposomes, useful in providing efficient transfer therapy.

Yet a further object of the present invention relates to providing amethod for long-term expression of a gene product from a non-integratedtransgene in a patient.

SUMMARY OF THE INVENTION

The present invention relates to liposome compositions and a method ofpreparing such liposomes. In addition, the present invention relates tothe administration of the biologically active agent-liposomepreparations to cells and further to the administration of the liposomepreparations to patients as a therapeutic agent.

The liposome compositions of the present invention provide highlyefficient delivery of biologically active agents to cells. Liposomevesicles are prepared from a mixture of a cationic lipopolyamine and aneutral lipid and form a bi- or multilamellar membrane structure(referred to herein as "DLS-liposomes") A preferred embodiment of thepresent invention uses a spermine-5-carboxy-glycinedioctadecylamide(referred to herein as "DOGS") as the cationic lipopolyamine anddioleylphosphatidyl ethanolamine (referred to herein as "DOPE") as theneutral lipid.

The liposomes of the present invention efficaciously deliverbiologically active agents into the cytoplasmic compartment of humancells. Use of such liposomal vehicles make possible high transfectionefficiency of biologically active materials into cells.

The present invention also encompasses a method of preparing such aliposome composition. The presence of at least one neutral lipid incombination with at least one cationic lipopolyamine makes possible theformation of liposomes after hydration. According to the method of thepresent invention, liposomes are prepared by mixing together each of acationic lipopolyamine and a neutral lipid in a molar ratio ranging froma ratio of 0.02:1 to a ratio of 2:1; evaporating the mixture to dryness;and rehydrating. In order to introduce a biologically active agent intothe liposomes, such agent can be added prior to or after rehydration ofthe dried film.

In one aspect of the invention, nucleic acids may be associated with theliposomes. This association may be accomplished in at least in two ways:(1) complex formation between the cationic liposome vesicle andnegatively charged polyaminon, such as nucleic acid or (2) encapsulationin the cationic liposome vesicle.

The present invention is further directed to a method of treating asubject with a suitable pharmaceutical formulation of nucleicacid-liposomes in order to deliver specific nucleic acids to targetcells of the subject. Such a method of treating subjects provideseffective delivery of oligonucleotides or gene-expressing nucleic acidvectors (e.g. plasmids or viral vectors) into cells. Therefore, such amethod of drug delivery is useful for the transport of nucleic acidbased therapeutics.

Another embodiment of the present invention is directed to a combinationof a DOGS/DOPE liposome preparation externally anchored throughhydrophobic interactions with an adenovirus particle. Since adenovirusesenter cells via receptor-mediated endocytosis, the combination ofadenovirus particles and the DLS-liposomes produces an enhancedtransduction efficiency.

Adenovirus particles may also serve as a nucleic acid which is carriedin the liposome internally.

The present invention provides a therapeutic method of treating ailmentsand conditions based upon a liposome-facilitated transfer ofbiologically active agents. For example, the present invention providesa pharmaceutical liposomal formulation for the delivery of nucleic acidsusing systemic administration to provide long-term expression of a givengene. One such method provides direct systematic nucleic acid transfercombining the DLS-liposomes with episomally replicative DNA vectorscarrying the nucleic acid of interest. Alternatively in vitro celltransfection followed by tissue transplantation such that thetransfected cells are incorporated in transplanted tissue. This methodis referred to as in vitro/ex vivo transfer. Other biologically activeagents may be encapsulated in the liposomes of the present invention anddelivered to cells using systemic or in vitro/ex vivo transfer methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: β-Galactosidase expression in HeLa cells transfected withDOGS/DOPE liposomes.

FIG. 2: Comparative study of the transfection efficiency of DLS andother liposomal delivery systems. Luciferase activity was assayed inHeLa cells transfected with pRSV-luc plasmid in presence or in absenceof serum (15%) T: DOGS or Transfectam™ (Promega, Madison, Wis.); LF:Lipofectin™ (Gibco BRL, Gaithersburg, Md.); LFA: Lipofectamine™ (GibcoBRL).

FIG. 3: Effect of preincubation in culture medium on DLS transfectionefficiency in HeLa cells. Luciferase activity was ascertained followingtransfection of liposomal DNA pre-incubated in serum containing medium(15%).

FIG. 4: Transfection efficiency in HeLa cells with liposomal DNA.Luciferase activity was determined.

FIG. 5: Relative transfection efficiency of DLS-liposome-1 andDLS-liposome-2 in HeLa cells using pRSV-luc.

FIGS. 6A-6D: Intracellular localization of FITC-end labeledoligodeoxynucleotides (20 mers) in HeLa cells following DLS treatment.Cells were exposed to 2 μM FITC-labeled oligodeoxynucleotides for 24 hr(A, B) and the post-incubated in drug-free medium for 24 hr (C). Cellswere treated with 2 μM free FITC-labeled oligodeoxynucleotides for 24 hr(D). Photographs represent computer-enhanced images from laser-assistedconfocal microscopy. Magnification, ×320.

FIG. 7: Protocol for liposomal transfection of mouse BMC.

FIG. 8: PCR of genomic DNA extracted from different hematopoietic cellstransfected in vitro/ex vivo and in vivo. Product size was assessed bycomparison with a 100-bp ladder run in parallel (upper row: lanes 1, 6,9; lower row: lanes 1, 4, 10). Upper row: lane 2 pHaMDR1 positive DNAcontrol; lane 3, genomic DNA from NIH3T3-MDR1 transfected, colchicineresistant cells; lane 4, DNA from NIH3T3 cells transfected with MDR1using the calcium phosphate precipitation protocol, but not selected indrug; lane 5, DNA from NIH3T3 parental cells; lane 7, BMC transfected 4days in vitro with DLS/MDR and selected in vincristine for 48 hours;lane 8, BMC transfected with DLS/Neo; lanes 10, 11, 12 show,respectively, BMC, spleen, and PB cells 15 days after treatment of amouse with i.v. injection of DLS/MDR1; lanes 13 and 14 show BMC andspleen cells from a mouse transplanted with BMC transfected in vitrowith DLS/MDR1, 15 days post-BMT. Lower row: lane 2, MDR1 positive BMCfrom a mouse transplanted 30 days earlier, lane 3, BMC from a controlDLS/Neo recipient mouse; lanes 5, 6, 7, BMC, spleen cells, and PB cellsrespectively from a DLS/Neo i.v. transfected mouse; lanes 8, 9, BMC, andspleen cells respectively from a transplanted mouse with DLS/Neotransfected BMC.

FIGS. 9A and 9B: FACSort analysis of BMC taken after in vitro DLS/MDRtransfection, before (A), and after (B) selection for 48 hours in 30ng/ml vincristine. The figure represents two histograms generated byFACSort analysis of BMC stained with MRK16 (plain line) or G2CL (dottedline), and GαM IgG-FITC. Displayed on the X axis is the fluorescenceintensity on a logarithmic scale, and on the Y axis is the relative cellnumber.

FIG. 10: Accumulation of rho123 after DLS/MDR, or DLS/Neo transfectionwithout selection. BMC transfected in vitro for 5 days with DLS/MDR(plain line) compared to DLS/Neo (dotted line) three hours afterexposure to rho123.

FIGS. 11A-11D: Appearance of CFU-Mix in methylcellulose obtained fromthe in vitro transfected BMC.

Representative photographs of CFU-Mix at day 12 of culture are shown.Panel A shows two colonies grown from control DLS/Neo transfected BMCwith no selection. Panel B shows two colonies grown from BMC transfectedBMC with DLS/MDR with no selection. Panel C, shows the absence ofcolonies from transfected BMC with DLS/Neo with 20 ng/ml vincristine,and panel D shows colonies of BMC transfected with DLS/MDR 20 ng/mlvincristine selection, and from transplanted and in vivo treated mice.

FIGS. 12A-12D: FACSort analysis of BMC taken from transplanted mice andfrom in vivo treated mice. The figure shows histograms generated byFACSort analysis wherein the X axis displays the fluorescence intensityon a logarithmic scale, and the Y axis displays the relative cellnumber. All histograms are generated after staining of BMC with MRK16and GαM IgG-FITC antibody. Panel A and B show, respectively, histogramsof BMC collected 15 days post-BMT and 25 days post-BMT. The recipient ofDLS/MDR transfected BMC is shown as plain line, and recipient of DLS/Neotransfected BMC as dotted line. Panel C and D show, respectively,histograms of BMC taken 12 and 25 days post-i.v. injection of DLS/cDNA.The plain line shows the DLS/MDR treated animal, and the dotted lineshows DLS/Neo transfected mice.

FIGS. 13A-13E: P-gp expression in BMC sub-populations taken fromtransplanted mice. The upper panels show the dot plot fluorescencerepresenting the cell size as forward side scatter (FSC) on the X axisand cell density as side scatter (SSC) on the Y axis. On the left,DLS/Neo, and right, DLS/MDR are shown cells transfected BMC taken frommice 15 days after BMT. The lower panels shown the histograms of thedifferent cell populations gated, and stained with MRK16 and GαMIgG-FITC. R1 shows the lymphocyte region, R2 shows the monocyte region,and R3 shows the granulocyte region. The staining of DLS/MDR transfectedcells is drawn as a plain line, and staining of DLS/Neo transfected BMCis shown as dotted line.

FIGS. 14A-14E: P-gp expression in BMC sub-populations taken from in vivotreated mice. The upper panel shows dot plot fluorescence: left,DLS/Neo, and right, DLS/MDR in vivo transfected BMC, 12 dayspost-injection. R1 shows the lymphocyte region, R2 shows the monocyteregion, and R3 shows the granulocyte region. MRK16 staining of thedifferent sub-populations of DLS/MDR transfected cells is drawn as aplain line, and staining of sub-populations of DLS/Neo transfected BMCare shown as a dotted line.

FIG. 15: Dose-response of luciferase DNA expression in different mousetissues. Increasing amounts of pBKd1RSV-luc plasmid DNA were injected inthe mouse tail vein and luciferase activity was determined 4 dayspost-injection in lung (▪), spleen () and liver (▴). All determinationsof luciferase activity were in triplicate and values are the means(±S.D.) from three different mice.

FIGS. 16A-16C: Effect of the route of administration on transgeneexpression. 25 μg pBKd1RSV-luc plasmid DNA were injected i.v., i.p. ands.c. Luciferase activity was assayed in liver, lung and spleen from twomice sacrificed 4 days after injection. Results are the means (±S.D.) oftriplicate luciferase activity determination from two different mice.

FIGS. 17A-17F: Immunohistochemical detection of luciferase in mice.Animals were treated with 75 μg pBKd2CMV-luc plasmid DNA delivered i.v.with DLS-liposomes-2 liposomes and sacrificed 6 days later.Immunostaining was performed using a horseradish peroxidase-coupledanti-rabbit antibody. Representative immunohistochemical stains onBalb/C mice liver tissue are presented: (A) heart; (B) liver; (D) colon;and (E) Kaposi's sarcoma cells implanted in immunodeficient mice. Thesections shown in (C) and (F) are respectively the sections shown in Band E treated with normal rabbit serum in place of anti-luciferaseantibody.

FIGS. 18A-18D: Time course of luciferase expression in differenttissues. 75 μg of pBKd2CMV-luc (closed bars) or pCMVintlux (open bars)plasmid DNA were administered i.v. and luciferase activity was assayedin lung, spleen, liver and heart up to 6 months post-injection. Valuesare the means (±S.D.) of triplicate determination from three differentmice.

FIG. 19: Luc expression in mouse tissues following i.v. liposomal DNAdelivery. Mice were sacrificed 2 weeks and 3 months after administrationof 75 μg DLS2-pBKd2CMV-luc. Luciferase mRNA was detected by using aRT-PCR technique on nucleic acids from frozen tissues.

FIGS. 20A-20B: Transmission Election Microseopy comparison. Panel Ashows DLS-liposomes and Panel B shows DOGS lipidic particles.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery that biologically activeagents may be associated with liposomes of a specific compositionforming a stable structure. The liposome containing a biologicallyactive agent may then be injected into a mammalian host to effectivelydeliver its contents to a target cell. One such example comprisesencapsulating a nucleic acid within a liposome and expressing a geneencoded on the nucleic acid within the target host cell, through the useof plasmid DNA. Conversely the expression of a gene may be inhibitedthrough the use of antisense oligonucleotides. Alternatively achemotherapeutic agent may act as the biologically active agent and beencapsulated within a liposome. The efficiency of a liposome-mediateddrug delivery system is directly dependent upon the liposome compositionand its resulting association with cellular membranes.

"Biologically active agents" as the term is used herein refers tomolecules which effect a biological system. These include molecules suchas proteins, nucleic acids, therapeutic agents, vitamins and theirderivatives, viral fractions, lipopolysaccharides, bacterial fractionsand hormones.

The term "protein" includes any proteaceous material such as peptides,protein fragments, protein conjugates, glycoproteins, proteoglycans,cytokines, hormones and growth factors.

The term "therapeutic agents" refers to any drug whose delivery could beaffected with liposomes. Therapeutic agents of particular interest arechemotherapeutic agents, which are used in the treatment and managementof cancer patients. Such molecules are generally characterized asantiproliferative agents, cytotoxic agents and immunosuppressive agentsand include molecules such as taxol, toxorubicin, daunorubicin,vinca-alcaloide, actinomycin and toposites.

The term "nucleic acids" means any double strand or single stranddeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) of variablelength. Nucleic acids include sense and anti-sense strands. Nucleic acidanalogs such as phosphorothioates, phosphoramidates, phosphonatesanalogs are also considered nucleic acids as that terms is used herein.Nucleic acids also include chromosomes and chromosomal fragments.

Antisense oligonucleotides may potentially be designed to specificallytarget genes and consequently inhibit their expression. In addition thisdelivery system may be a suitable carrier for other gene-targetingoligonucleotides such as ribozymes, triple helix formingoligonucleotides or oligonucleotides exhibiting non-sequence specificbinding to a particular proteins of other intracellular molecules. Forexample, the genes of interest may include retroviral or viral genes,drug resistance genes, oncogenes, genes involved in the inflammatoryresponse, cellular adhesion genes, hormone genes, abnormallyoverexpressed genes involved in gene regulation.

Such nucleic acids may be associated with a liposome composition inaccordance with this invention. In one embodiment of the presentinvention, modified or unmodified phosphodiester oligonucleotides(alternatively referred to as "oligo(dN)") are used as nucleic acids.These analogs provide increased nuclease protection and increasedcellular transport.

"Liposome" as the term is used herein refers to a closed structurecomprising of an outer lipid bi- or multi-layer membrane surrounding aninternal aqueous space. Liposomes can be used to package anybiologically active agent for delivery to cells. For example, DNA can bepackaged into liposomes even in the case of plasmids or viral vectors oflarge size which could potentially be maintained in a soluble form. Suchliposome encapsulated DNA is ideally suited for direct application to invivo systems by a simple intravenous injection. These liposomes mayentrap compounds varying in polarity and solubility in water and othersolvents. The liposomes are generally from a bilayer membrane in a uni-or multilamellar membranous structure. Generally these liposomes mayform hexagonal structures, and suspension of multilamellar vesicles.

The lipid mixture of the present invention comprises a cationiclipopolyamine compound. Cationic lipopolyamines useful in the presentinvention include cationic lipid derivatives of polyamines, spermidineand spermine as well as others well-known in the art (Theoharides, 1980Life Sci. 27:703-713; Stevens, 1981 Med. Biol. 59:308-313; Morris,Marton Eds., Polyamines in Biology & Med., Dekker, N.Y., N.Y., p. 512;U.S. Pat. No. 5,171,678 and U.S. Pat. No. 5,283,185, all of which areincorporated herein by reference). Examples of cationic lipopolyaminesinclude2,3-dioleyloxy-N[2sperminecartoxamido)ethyl]-N,N-dimethyl-1-propanaminuimtrifluoracetate, spermine-5-carboxy-glycinediotadecylamide anddipalmitoylphosphatidylethanolamidospermine. A preferred type oflipopolyamine comprises a lipid having a quaternary or tertiary aminegroup covalently attached.

These lipopolyamines can vary in chain length and may be present asmixtures of lipopolyamines in the liposome so long as the molar ratio oflipopolyamine to neutral lipid is maintained.

A preferred cationic lipopolyamine is DOGS.

In order to form stable liposomes, the cationic lipopolyamine iscombined with a neutral lipid. Such neutral lipids includetriglycerides, diglycerides and cholesterol and are known in the art,for example as described in U.S. Pat. No. 5,438,044 which isincorporated herein by reference. In particular a neutral phospholipidis preferred. More preferably, the neutral lipid is a neutral aminophospholipid. Most preferably, the neutral lipid comprisesphosphatidylethanolamine (PE) or a derivative of PE such as DOPE(dioleoyl-phosphatidylethanolamine). Mixtures of neutral lipids may beused in the liposomes of the present invention so long as the molarratio of lipopolyamine to neutral lipid is maintained.

Liposomes comprising at least one lipopolyamine and at least one neutrallipid, present in a molar ratio range of 0.02:1.0 to a ratio of 2.0:1.0provide an effective drug delivery system. More preferably, the molarratio of lipopolyamine to neutral lipid is 0.2:1 to a ratio of 0.9:1.For optimal transfection efficiency a cationic lipopolyamine/neutrallipid molar ratio of about 0.5:1 is used. The liposomal composition ofthe present invention has shown to be stable in a biologicalenvironment. In the case wherein a nucleic acids is the biologicallyactive agent, it is demonstrated that nucleic acids associated with theliposomal carrier are completely protected from enzymatic attack, suchas from nucleases, and that stability in circulating blood afteradministration may be achieved.

The liposome delivery system of the present invention comprises aspecific mixture of lipids. These components are prepared such that aliposome is formed and the biologically active agent, such as a nucleicacid is contained therein.

Nucleic acids-based therapeutics are of broad use in therapy of a widevariety of diseases and disorders, such as, inherited or acquiredgenetic disease or viral infections. In addition, nucleic acid basedtherapeutics can be used to prevent drug resistance.

The present invention may utilize one or more nucleic acids or otherbiologically active molecules in conjunction with the liposomal carrier.

Method of Preparing DLS-Liposomes

In one embodiment of the present invention, the liposomes are preparedby drying a lipid mixture containing a cationic lipopolyamine andneutral lipid which are provided preferably in a molar ratio range of0.02:1 to a ratio of 2.0:1. This dried film is then rehydrated. Severalmethods of associating biologically active agents, for example nucleicacids, with liposomes are described in this invention. The exemplifiedembodiment comprises hydrating a dried lipid film by introducing anaqueous solution, and completely dispersing it by strongly homogenizingthe mixture with a vortex, magnetic stirrer and/or sonication.Subsequent liposomes are mixed with a nucleic acid solution allowingcomplex formation between positive charges of thelipopolyamine-containing liposomes and the negative charges of thenucleic acids. Such liposomes are referred to herein as DLS-liposome-1or lipid complexes.

In another embodiment of the present invention the liposomes are formedby dissolving in chloroform at least one cationic lipopolyamine and atleast one neutral lipid. After stirring by gentle vortexing, the mixtureis evaporated to dryness. The subsequent dried lipid film is resuspendedin a volume of water containing the biologically active agent. Formationof DLS-liposome is carried out by thorough stirring. Entrapment and/orassimilation of the biologically active agent by the DLS-liposomes isefficient and nearly complete.

The exemplified embodiment comprises hydrating the dried lipid filmusing a low and defined (5-10 λl/μg lipids) volume of aqueous solutioncontaining concentrated nucleic acids. These liposomes are referred toherein as DLS-liposomes-2 or encapsulated lipsomes. Such concentratednucleic acids are provided in a concentration greater than 1 mg/ml. Apreferred concentration is about 2 mg/ml and the most concentrated formof nucleic acid will depend upon the concentration at which itsviscosity is excessive, generally at a concentration of about 3 mg/ml.Dispersion is completed by strongly homogenizing the mixture using avortex or magnetic stirrer. Nucleic acids are encapsulated in theliposomes during the formation and also are partly complexed throughelectrostatic interaction between the nucleic acid and the cationicliposomes.

In another embodiment of the present invention other biologically activeagents are encapsulated in a DLS-liposome. The second method describedabove can be used with any biologically active material. Therefore,molecules such as chemotherapeutic agents can be introduced into theliposomes of the present invention by rehydrating the dried film in thepresence of such agents.

The methods of forming liposomes of the present invention lead toliposome-complexed and liposome-encapsulated biologically active agents.Liposome-encapsulated biologically active agents have been shown to bemore efficient in transducing cells in cell cultures. However, theability to sonicate the lipid vesicles in the liposome-complexedbiologically active agents allow for more homogenized and smallerliposome particles, and consequently for the ability to circulate forlonger periods in blood following systemic injection.

Biologically active agents delivered using the delivery system of thepresent invention are efficiently released from endocytic vesicles, andas a result, a high cytoplasmic and nuclear distribution of biologicallyactive agents is achieved.

Targeted Liposomes

The presence of a neutral lipid, such as DOPE, in combination with acationic lipopolyamine such as DOGS makes possible the formation ofliposomes upon rehydration, whereas use of a lipopolyamine alone onlyleads to the formation of lipid particles. Formation of phospholipidicbilayer or multilamellar membrane vesicles (liposomes) allows for aenhanced blood circulation, stability and effectiveness of cellularuptake. In addition, the liposomal membrane facilitates anchorage to itssurface of other substituents, which can increase gene transfer andallow cell targeting, such as viral particles, virus fusogenic peptidesspecific ligands or antibodies.

Formation of liposomes make possible anchorage to the membrane layers ofproducts which may increase transduction efficiency. Viruses, ingeneral, are inherently excellent gene transfer vectors. Viral capsidsor envelopes exhibit specific structure and contain molecules leading toefficient delivery of their genetic content to the infected cells. Inorder to exploit these properties, adenovirus particles (without DNA ordenatured by irradiation) have been externally attached to the liposomalmembrane of the liposomes prepared according to the present invention.It is established that adenoviruses enter cells via receptor-mediatedendocytosis. A specific fusogenic mechanism makes possible the releaseof the viral genetic content from the cellular endocytic vesicles afterinternalization. Use of adenovirus to facilitate gene transfer has beenreported (Cristiano R. J., et al. Proc. Natl. Acad. Sci. USA 90,2122-2126 (1993)). Although DLS-liposomes clearly show a significantescape from intracellular vesicles, presence of adenovirus capsids atthe liposome surface may enhance transduction efficiency by facilitatingintracellular vesicle disruption.

Alternatively, tissue targeting may be obtained by anchoring antibodiesor ligands at the surface of the liposomes. Cell specificity of suchliposome mediated delivery may be of particular importance in targetingcancer cells and bone marrow stem cells.

The liposomal delivery system of the present invention may be used forincreasing recombinant retrovirus and adenovirus infection. Retrovirusentry into cells is mediated via ligand-receptor recognition, andconsequently their uptake is very low in certain cells which do notpresent those receptors. Associating a retrovirus or other recombinantvirus such as adenovirus to be used for gene therapy onto the outside ofthe liposomes may enhance penetration and/or expression of the viralagents.

The liposomal delivery system of the present invention makes possiblehigh transduction efficiency in any type of cell, including humanadenocarcinoma, HeLa, murine carcinoma, NIH3T3, human embryonic kidney293, human leukemia MOLT-3 cell lines, and primary cultures of humanmacrophages and human vascular endothelial cells.

Transfer Therapy Methods

The liposomal composition of the present invention may be systematicallyadministered into patients parenterally in order to achieve transfertherapy of one or more biologically active agents. Moreover, thistechnique may be used for "ex vivo" transfer therapy where tissue orcells are removed from patients, then treated and finally reimplanted inthe patient. Alternatively, systemic therapy is also effective inadministering the DLS-liposome.

Many diseases can be treated via the drug delivery system of the presentinvention. Diseases such as diabetes, atherosclerosis,chemotherapy-induced multi-drug resistance, and generally,immunological, neurological and viral diseases can be treated using thepresent drug delivery system. One particular condition which can betreated via the system of the present invention relates to HIV andHIV-related diseases, such as anemia, leukopenia and thrombocytopenia.These clinical conditions are significantly related to a decrease ordisappearance of hematopoietic progenitor cells in bone marrow of HIV-1patients. Transfection of bone marrow stem cells, bone marrow stromacells and embryonic stem with gene coding for immuno-restoring compoundsmight enhance the differentiation and proliferation capacity of suchcells.

The delivery system of the present invention is also useful forcorrecting the ion transport defect in cystic fibrosis patients byinserting the human CFTR (cystic fibrosis transmembrane conductanceregulator) gene. Oral administration such as nebulization couldparticularly suitable. In addition, DLS-liposomes can be used for theinhibition of tumor cells by administering in tumor cells a moleculeinhibiting tumorigenesis or gene coding for an antisenseoligonucleotides directed to mRNA transcripts of angiogenic factors. Inaddition, ribozymes may be encapsulated and enzymatically attachspecific cellular contents. Intra-lesional or intravenous administrationappear suitable for this case.

The ability to select bone marrow cells expressing a selectable genewhich confers resistance to anti-cancer drugs would be useful to protectbone marrow during chemotherapy, but also could be helpful to select forcells co-transfected with genes needed in therapy of other diseases,including genetic defects manifested in bone marrow. One such selectablegene is the human MDR1 gene which confers cross-resistance to manycytotoxic drugs. The human MDR1 gene product is a 170 Kd glycoprotein(referred to herein as "P-gp") that works as an ATP dependent pump thateffectively pumps out of cells many anti-cancer cytotoxic drugs, such astopside, teniposide, actinomycin D, doxorubicin, daunorubicin, taxol, orvinca alkaloids.

An exemplified embodiment of the present invention describes anefficient protocol for introducing the human MDR1 gene intohematopoietic cells both in vivo and in vivo using a liposomal deliverysystem. Transfection of hematopoietic cells followed by gene expressionis demonstrated in at least three blood cell lineages.

Using the DLS-liposome system, the human MDR1 gene is introduced intobone marrow cells ("BMC"). The transferred human MDR1 is expressed, asdetected by staining with P-gp specific MRK16 monoclonal antibody, inall of the in vitro transfected BMC. Moreover, P-gp is detected in BMCfrom all transplanted animals tested, and from almost all of the in vivotreated animals.

The expression of the MDR1 gene appears to be present for a period of atleast 30-36 days, indicating that some of the transfected cells had beenprecursor cells, or long-lasting cells.

The potential for obtaining drug resistant bone marrow progenitor cellsafter gene transfer using the instant liposome delivery system make itpossible to protect cancer patients undergoing chemotherapy from marrowtoxicity of anti-cancer drugs. In addition, the multidrug resistancegene serves as a positive selectable gene marker in vivo for insuringthe expression of a non-selectable gene.

Alternatively a systemic approach to transfer therapy may be utilized.

The DLS-liposomes containing the nucleic acid drug can be administeredby intravenous, intramuscular, intraperitoneal, subcutaneousintra-lesional and oral means.

The development of the present liposome delivery system comprising DLSliposomes may be encapsulate episomal expression vectors so as to resultin a broad biodistribution and persistence of transgene expressionfollowing a single intravenous ("i.v.") injection of liposomal DNA. Theefficacy of DLS-liposomes used for the "in vivo" expression of the humanMDR-1 gene is also disclosed in bone marrow progenitor cells byemploying two different approaches: 1) a systemic delivery, and 2) an"ex vivo" approach by transplanting "in vitro" transfected BMC.

The long-term transgene expression observed using these delivery methodsis due to the ability of DLS-liposomes to deliver significant amounts ofDNA in to cells and tissues and to the use of human papovavirus("BKV")-derived episomal expression vectors. Episomal vectors may bederived from the BKV contain a viral origin of DNA replication and aviral early gene that transactivates the viral DNA origin ofreplication, allowing for episomal replication in permissive cells.BKV-derived expression vector share the desirable qualities ofextrachromosomal replication and thus a lack of a requirement ofcellular division for the chromosomal replication of retroviral-basedvectors. Moreover, extrachromosomal expression may lessen thepossibility of attenuation of the transgene expression due to hostcis-chromosomal effects. The BKV-derived episomes may persist in theprogeny of transfected cells, whereas non-episomal vectors would notpersist in a non-integrated form following cell division. Other episomalexpression vectors include as Epstein-Barr virus derived vectors (Yates,J. L. et al. (1985) Nature 313, 812-815) and can also be used for "invivo" gene transfer therapies.

Episomal expression constructs utilized in the present invention improvethe persistence of transgene expression when compared with the use ofnon-episomal vectors. Using PCR analysis, detection of the transgene invarious organs is possible and detection of the transgene mRNA persistsfor as long as 3 months. Transgene expression of the present inventiondeclines slowly after a maximum level between 6 and 15 dayspost-injection and is detected for up to 3 months in various tissues.Original and episomally replicated forms of BKV-derived derived vectorsare present in tissues 2 weeks post-injection suggesting episomalreplication from this point on.

Episomal DNA vectors in combination with an efficient synthetic deliverysystem appears to be a particularly attractive approach for genetransfer therapy. The present invention designed BKV-derived episomalconstructs which lack the expression of viral proteins (VP1, VP2 andVP3) so as to avoid the side effects associated with the expression ofviral proteins. These constructs (pBKd2) retained high transfectionefficiency and "in vivo" episomal replication.

A degree of tissue specific expression can be obtained depending uponthe liposome preparation, the route of administration and the promoterdriving expression of the transgene. Useful promoters are well-known tothe skilled artisan and can be substantiated for those exemplifiedherein. It is clear that the more cationic (DNA/total lipid ratio <0.05,w/w) the liposomes, the more lung and heart are targeted. Althoughreporter gene expression may be lower following subcutaneous ("s.c.")administration of liposomal DNA compared to i.v. administration, therewere no changes in tissue targeting. In contrast and as expected, afterintraperitoneal ("i.p.") administration the spleen was particularlytargeted. The present invention also demonstrates that the CMV promoteris capable of more efficient expression in spleen than in lung whencompared to the RSV promoter. No significant difference has beenobserved in liver and heart. Thus, the choice of the promoter maygreatly influence the efficacy of non-retroviral mediated gene deliveryand may lead to a certain degree of tissue specificity.

In one embodiment of the present invention, the transgene expressionfollowing a single injection of liposomal DNA was investigated. It isclear that repeated injection may increase and/or prolong transgeneexpression. Then, desirable transgenes may be repetitively administratedand thus offers an attractive alternatives to retroviral mediated genetherapy. Using the DLS-liposomes of the present invention administeredvia systemic delivery or aerosol delivery induced immunogenecity wasobserved when liposomal DNA was administrated at doses which produceddetectable transgene expression.

A proposed daily dosage of active compound for the treatment of man is0.5 mg DNA/kg to 4 mg DNA/kg, which may be conveniently administered inone or two doses. The precise dose employed will of course depend on theage and conditions of the patient and on the route of administration.Thus a suitable dose for administration by inhalation is 0.5 mg DNA/kgto 2 mg DNA/kg, for oral administration is 2 mg DNA/kg to 5 mg DNA/kg,for parenteral administration is 2 mg DNA/kg to 4 mg DNA/kg.

The compound of the invention may be formulated for parenteraladministration by bolus injection or continuous infusion. Formulationfor injection may be presented in unit dosage form in ampoules, or inmulti-dose containers with an added preservative. The compositions maytake such forms as suspension, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for reconstitution with a suitablevehicle, e.g. sterile pyrogen-free water, before use.

The compounds according to the invention may be formulated foradministration in any convenient way. The invention therefore includeswithin its scope pharmaceutical compositions comprising at least oneliposomal compound formulated for use in human or veterinary medicine.Such compositions may be presented for use with physiologicallyacceptable carriers or excipients, optionally with supplementarymedicinal agents. Conventional carriers can also be used with thepresent invention.

For oral administration, the pharmaceutical composition may take theform of, for example, tablets, capsules, powders, solutions, syrups orsuspensions prepared by conventional means with acceptable excipients.

The following examples serve to illustrate further the present inventionand are not to be construed as limiting its scope in any way.

All of the references mentioned in the present application areincorporated in toto into this application by reference thereto.

EXAMPLE 1

Preparation of a DOGS/DOPE liposome composition.

Liposomes are formed by mixing 1 mg DOGS and 1 mg DOPE (0.5:1 molarratio). After thorough stirring, the mixture is evaporated to dryness ina round bottomed borosilicate tube using a rotary evaporator. Thesubsequent dried lipid film is resuspended in a low volume of ethanol(10 to 40 pl/mg lipid). Formation of liposomes is carried out by addingan excess of distillated water (at least 200 μl/mg lipid). Afterhomogenization by slight vortexing, the mixture is incubated for atleast 15 min. If needed, the resulting suspension may be sonicated in afixed temperature bath at 25° C. for 15 min.

EXAMPLE 2

Preparation of a DOGS/DOPE liposome-nucleic acids complex composition.

Complex formation of nucleic acids to the liposome bilayer membrane isachieved by simply mixing the preformed DOGS/DOPE liposomes to asolution of nucleic acids. In an Eppendorf tube, DLS-liposomes are mixedin a 150 mM NaCl solution to nucleic acids at a concentration of 12.5 μgtotal lipids (liposomes)/μg nucleic acid for double strand DNA, and aconcentration of 6 μg liposomes/1 μg nucleic acid for oligonucleotides.The mixture is slightly mixed and incubated for at least 30 min at roomtemperature. Complex formation is very effective and nearly completesince at least 80% of nucleic acids were assimilated into the liposomes.These liposomes are referred to as DLS-liposomes-1 or liposomecomplexes.

EXAMPLE 3

Alternatively, liposomes were formed by mixing 1 mg DOGS and 1 mg DOPE(0.5:1, molar ratio). After thorough stirring, the mixture is evaporatedto dryness in a round bottomed borosilicate tube using a rotaryevaporator. The subsequent dried lipid film is resuspended in a minimalvolume (7 μl/mg lipid) of water solution containing nucleic acids (1400μg/ml). Formation of liposomes is carried out by thorough stirring. Thesubsequent liposome preparation may be diluted in 150 mM NaCl.Entrapment and/or assimilation of nucleic acid by the liposomes is veryefficient and nearly complete since at least 80% of nucleic acids areencapsulated by the liposomes. These liposomes are referred to asDLS-liposomes-2 or encapsulated liposomes.

EXAMPLE 4 In vitro Gene Transfection

Gene transfection efficacy was ascertained in vitro using reporter genessuch as genes coding for β-galactosidase or luciferase. Two plasmidconstructs containing the CMV (Clonetech) and RSV (Promega) promoterswere used as genetic vectors for the β-galactosidase and luciferasegenes, respectively. These plasmid vectors were delivered to the humancarcinoma HeLa cells via the liposome carrier system in accordance withthis invention. Either DLS-liposmes-1 or DLS-liposomes-2 are used inthis example.

One microgram liposomal DNA (both DLS-liposmes-1 and 2) was added to themedium of a HeLa cell culture at a 50-700% confluency (500,000-700,000cells/ml culture medium/7 cm² culture plate surface area). Cells wereincubated at 37° C. with the liposomal DNA for at least 4 hr.Determination of gene expression was carried out for both types ofplasmids following an incubation of 2-3 days at 37° C.

β-galactosidase activity was observed using a staining procedure aftercell fixation on the culture plate using a conventional method. Cellsexpressing the β-galactosidase were readily identified by their intenseblue staining. As shown in FIG. 1, more than 60% of the HeLa cellstreated by DOGS/DOPE liposomal DNA, actively expressed β-galactosidase.

Luciferase activity was detected in HeLa cells using a standard method(Promega, Madison, Wis.). Luciferase gene containing plasmid was usedfor a comparative study of the transduction efficiency of the liposomaldelivery in accordance with this invention and liposomal vectorscommercially available. Optimal experimental conditions were used foreach tested method.

In serum-containing cell culture medium, transduction efficiency in HeLacells treated with the liposomal system in accordance with thisinvention appears to be 11-fold, 10-fold and 37-fold higher than that ofDOGS or Transfecta™ (Promega, Madison, Wis.), Lipofectin™ andLipofectamine™ (Gibco BRL, Gaithersburg), as shown in FIG. 2.

In serum-free medium, transduction efficiency using DLS-liposomesappears equivalent to that determined when cells are incubated inserum-containing medium. In contrast, use of Lipofectamine™ inserum-free conditions make possible a high transfection efficiency. Thedramatic decrease in transduction efficiency (186-fold) usingLipofectamine™ in a medium containing fetal bovine serum (10%)emphasizes the high instability of DNA when exposed to nucleases, andthe need for complete DNA protection from enzymatic attack in abiological environment. DOGS/DOPE liposomes prepared in accordance withthe method described in example 2 and 3 exhibit an effective celldelivery and an efficient DNA protection during transport.

As illustrated in FIG. 3, preincubation of liposomal DNA in serumcontaining medium up to 48 hours does not decrease transfectionefficiency.

FIG. 4 shows optimal transfection efficiency by using a 12:1liposome/DNA weight ratio for the preparation of the liposomal DNA.

As illustrated in FIG. 5, use of plasmid DNA delivered byDLS-liposomes-2 showed better transduction efficiency in HeLa cells whencompared to the DLS-liposomes-1 delivery. This may be due to increase inDNA protection from nuclease attack and/or better release from endocyticvesicles when encapsulated in liposomes.

EXAMPLE 5 Preparation of DOGS/DOPE Liposome Composition ContainingOligonucleotides

Oligonucleotides may be complexed or encapsulated in DLS-liposomes usingthe method described in example 2, respectively. The only modificationis that a two-fold higher nucleic acid/liposome ratio is preferably usedfor DLS-liposomes-1 (20 μg/60 μg, weight ratio) to produce an equivalentcomplexing efficiency. This leads to a higher concentration ofoligonucleotides complexed with the liposome preparation andconsequently a higher efficiency of delivery in terms of quantity ofnucleic acids delivered per cell.

EXAMPLE 6 Intracellular Distribution of Nucleic Acid After Delivery withDLS-Liposomes

Nucleic acid cell penetration and its intracellular distributionfollowing delivery using DLS-liposomes were observed usinglaser-assisted confocal microscopy and FITC-labeledoligodeoxyribonucleotides (20 mers). The former technique allows for thehigh resolution of optical sections of suspension cell preparations andcan readily specify the intracellular distribution of a fluorescentcompound. FIG. 6 presents images of hepatocyte HepG2 cells treated withDLS-1 encapsulated oligodeoxyribonucleotides for 24 hrs.

A high penetration of the labeled oligodeoxyribonucleotides was observedin all intracellular compartments (FIG. 6A). In order to investigatewhere oligodeoxyribonucleotides are highly concentrated, wesignificantly reduced the gain of the laser beam used for confocalmicroscopy observation of the same cell, and observed a punctuatedintra-cytoplasmic distribution of the oligonucleotides (FIG. 6B). Thissuggests that DLS-liposomes transport oligonucleotides into cells viaendocytosis and then oligonucleotides quickly escape from endocyticvesicles leading to a release of free oligonucleotides in the cytoplasm.Oligonucleotides are immediately transported from the cytoplasm to thenucleus (FIG. 6C). An extremely weak fluorescence intensity was observedin cells incubated with free labeled oligonucleotides, suggesting poorpenetration and/or degradation by nucleases present in theserum-containing culture medium (FIG. 6D).

This observation is of great interest since it shows efficient deliveryof nucleic acids to cells, total and immediate escape from endocyticvesicles where active degradation could take place, and nuclearlocalization after cell treatment. Theoretically, cell delivery ofplasmid-DNA via DLS-liposomes may use the same pathway of cellinternalization.

EXAMPLE 7 Preparation of a DOGS/DOPE Containing Liposome Compositionwith Adenovirus Particles

The adenovirus strain used in the present invention is the dl 312 strainreceived as a gift from T. Shenk (Princeton Univ., Princeton, N.J.). Anyadenovirus strain can be used in the present invention. Preparation ofadenovirus capsids (no DNA) using cesium chloride gradient method may beperformed following adenovirus collection and preparation. Wholeadenovirus particles (with DNA) but inactivated by UV irradiation (10J.m⁻² 8-1) may also be used. Hydrophobic binding of adenovirus to theliposomes is carried out by simply mixing the liposome suspension withthe adenovirus concentrate. For example, particles equivalent to 10⁸ PFU(Plating Forming Unit) are added to 12.5 μl of a DLS-liposome-associatedDNA preparation corresponding to 1 μg DNA and 12.5 μg lipids. Themixture is slightly homogenized and then incubated at 37° C. for 1 hrwith gentle shaking. Immediately after incubation adenovirus-DNAliposome preparation is added to cell culture. Transfection procedure isthe same as that used for DLS liposomes.

FIG. 7 illustrates that adenovirus associated liposomes greatly enhancetransfection efficiency in HeLa cells by a factor 4.5. Transfectionefficiency obtained after simultaneous addition in cell culture mediumof liposomal DNA and adenovirus particles at equivalent concentrationwas significantly lower (2.7-fold). This demonstrates the specificadditional effect of the adenovirus particle attachment to the liposomeson gene expression and particularly on the plasmid DNA escape fromendocytic vesicles.

EXAMPLE 8 Transmission Electron Microscopy Comparison of LipidicParticles and DLS-Liposomes

DLS-liposomes and Transfectam™ (Promega, WI) reagent (DOGS) samples weresubmitted to negative staining and Transmission Electron Microscopy(TEM) analysis. The following is a part of the observationsindependently made by ABI Inc (Columbia, Md.).

DLS-liposomes: "Lipidic particles were observed throughout thispreparation and were found in large quantities." "Each differentparticle appeared to display a heavily stained core region, which wassurrounded by many different layers of membranes or envelopes. Theparticles contained so many different layers of membranes, it wasdifficult to establish the size of one lipidic particle to the next.Although it was difficult to measure the overall size of the particles,due to their pleomorphic shape and varied number of layers, it appearedthe particles ranged from 200 to 3000 nm in diameter. The grid areasshowed a high concentration of smaller lipidic particles throughout thebackground of the sample".

Transfectam™ reagent (DOGS): "Possible lipidic particles were found inthis sample, in small quantities. The particles found in this samplewere very different from those observed in the previous sample. Thelipidic particles observed appeared to be either in the process ofbreaking down or they had never properly been formed. Large areas oflipid-like material were observed, however, they did not display anyultrastructural detail, such as different layers of membranes. The onlysimilarity between this sample and the previous sample was that thelipidic particles were heavily stained. Very little debris was found inthe background of the sample."

Thus, TEM analysis as demonstrated that DLS-liposomes are bilayermembranes vehicles. This specific ultrastructure differentiatesDLS-liposomes from the Transfectam™ reagent or other cationic liposomesthus far commercialized, such as Lipofectin™ (BRL Co., ND). Thesenonliposome particles, when complexed with DNA do not form membranebilayer-containing vesicles but rather are lipid coating particles thatpresumably contain nucleic acids. Thus they are not be liposomes in thetrue sense of the term. DLS-liposomes provide better efficacy intransferring DNA which can be explained by their liposomal structure.Furthermore, we may expect improved pharmacokinetic properties such asincreased plasmid half life. In addition, the presence of a membranebilayer in DLS-liposomes makes possible the anchorage of antibody totheir surface which may result in cell targeting.

EXAMPLE 9 Expression of the MDR-1 Gene in Cultured Murine Bone MarrowCells

The MDR-1 gene expresses the P-glycoprotein ("P-gp"), a plasma membraneprotein involved in the emergence of the Multi-drug Resistance phenotypewhich may occur after chemotherapy. The MDR-1 gene was used in thisexample as a marker of gene delivery in order to assess the efficacy ofbone marrow transplant of MDR-1 gene transfected bone marrow cells byDLS-liposomes, both DLS-1 and DLS-2.

In order to assess the efficacy of bone marrow transplantation for "exvivo" gene therapy, murine bone marrow cells were transfected with thisplasmid and the DLS-liposomes and transplanted into Balb-C mice. Theproliferation and differentiation of transduced hematopoietic progenitorcells were detected up to 21 days after transplantation in the spleenand the bone marrow, suggesting that the bone marrow transplant hadtaken place.

Murine bone marrow cells were harvested and quickly transfected with thepHaMDR GA plasmid encapsulated in DLS-liposomes. Seven differentexperiments have confirmed that the MDR-1 gene was expressed in bonemarrow cells since cells continue to grow under selective pressures(vincristine). In addition, lymphocyte, macrophage and fibroblastpopulations have been shown to exhibit the MDR phenotype after selection(using the rhodamine drug efflux method).

EXAMPLE 10 In vitro and in vivo Transfection of BMC

In order to achieve efficient liposomal transfection, both ex vivo andin vivo approaches have been used. The protocols shown in FIG. 1 wereutilized. In examples 10-13, DLS-liposomes-2 are used. 1) the invitro/ex vivo approach, in which mice were pre-treated with 5-fluoroUracil ("5-FU") (150 mg/kg) by the method described in Hodgson et al.(1979 Nature 281:381-2) were sacrificed, and their bone marrow cells("BMC") were transfected with 10 μg of DLS/MDR in T25 culture flasks(Costar). BMC transfected with DLS/Neo was used as negative controls.After 4-5 days, BMC were transplanted into lethally irradiated mice.Some BMC were kept for analysis by FACSort, PCR or kept in suspensionculture with or without vincristine for 48 hours after which they weretested in semisolid medium for their potency to form colonies. 2) Thedirect in vivo gene delivery approach was used, in which 2-3 days afterbeing pre-treatment with 5-FU (150 mg/kg), mice were injectedintravenously with 75 μg DLS/MDR. All negative control mice wereinjected with DLS/Neo.

Mice from both groups were sacrificed at different time points, andhematopoietic cells were collected for analysis by PCR, FACSort, orassayed in methylcellulose for the potential to form colonies in thepresence of different concentrations of vincristine.

Mouse peripheral blood (PB) was obtained by eye bleeding. Mouse BMC wereobtained by flushing the long bones with DMEM using a 21 gauge needle.Spleen cells were obtained by pressing the spleens with the barrel of a3 cc syringe.

Bone marrow transplantation ("BMT") procedure

To assess functional expression of P-gp, 1×10⁶ transfected cells andcontrol cells were incubated in FACSort medium containing 1 μg/mlrhodamine 123 (rho123) (Sigma) at 37° C. for 15 minutes. After washing,the cells were transferred into rho123 free medium at 37° C., andincubated for 3-4 hours. The cells were then washed and analyzed byFACSort. Results were displayed as histograms, where efflux of rho123would be registered as a decease of fluorescence intensity.

Mouse Bone Marrow Cell Culture

Bone marrow cells (BMC) were harvested from 6-12 weeks old C57B1/6 micepurchased from Frederick Research Laboratories (Frederick, Md.), andhoused in a specific pathogen-free environment. Mouse BMC culture wascarried out in DMEM supplemented with 50 μg/ml penicillin/50 μg/mlstreptomycin, 2 mM glutamine (Gibco laboratories, Greenbelt, Md.), and10% calf serum (Colorado Serum Company, Denver, Colo.). Cell growthfactors mouse II3 9100 ng/ml), human II6 (200 ng/ml) (CollaborativeScience Inc.), and rat SCF (10 μg/ml) (generously provided by Amgen, SanDiego, Calif.) were added to the media before each experiment.

Plasmids

The MDR1 retroviral expression plasmid, pHaMDR1/A containing wild typehuman MDR1 cDNA under transcriptional control of the Harvey MurineSarcoma Virus-Long Terminal Repeat (Ha-MSV-LTR) sequences has beendescribed (Pastan, et al. 1985 PNAS 85:4486-90). A plasmid containingthe neomycin resistance gene wit the same promoter sequence as pHaMDR1/Awas used as negative control (pHaNeo) (Zhu et al. 1993 Science261:209-11).

Liposomes were formed by dissolving in chloroform 1 mgspermine-5-carboxy-glycinedioctadecylamid (DOGS) and 1 mg of the neutrallipid DOPE (0.5:1, molar ratio) designated further as DLS. Afterstirring by gentle vortexing, the mixture was evaporated to dryness. Thesubsequent dried lipid film was resuspended in a minimal volume (7 ml/mglipid) of water containing plasmid (1.4 mg/ml). Formulation of DLS wascarried out by thorough stirring. Entrapment and/or assimilation of theplasmid by the DLS is efficient and nearly complete. pHaMDR1/A, andpHaNeo plasmids entrapped in DLS were identified respectively asDLS/MDR, and DLS/Neo.

Polymerase Chain Reaction

Total genomic DNA from in vitro transfected BMC, as well from differenthematopoietic cells collected from transplanted and in vivo transfectedmice, was obtained using a DNA extract kit (Gentra systems, Inc.,Research Triangle Parc, N.C.). The DNA yield and purity were tested byUV spectroscopy. PCR was carried out with 1 μg total DNA, 1 unit ofAmpliTaq Polymerase and reaction kits (Perkins Elmer, Roche, Branchburg,N.J.) in a final volume of 100 μl. Each cycle of PCR included a 1 minutestep of denaturation at 95° C., a 1 minute step of primer annealing at57° C., and a 1 minute step of extension/synthesis at 72° C. Thepresence of human MDR1 gene specific sequence were probed by using a setof primers described in Noonan et al. (1990 PNAS 87:7160-4) yielding aproduct size of 167 bp. Each primer was added at 40 pmol per reaction.PCR products were separated on a 2% agarose gel and stained withethidium bromide.

Selection of BMC in suspension culture

3×10⁶ BMC were added to BMC culture medium containing 30 ng/mlvincristine (Sigma). Cells were left for 48 hours in culture after whichfloating cells were collected and pooled with adhering mouse BMC thatwere detached using cell scrappers. Cell were counted on a hematocymeterand viability was established by trypan blue exclusion.

Transfer of MDR1 in gene BMC after transfection using DLS Liposomes

To assess the transfer of the human MDR1 gene in DLS/MDR transfectedmurine cells, genomic DNAs from different NIH3T3 and hematopoietic cellswere tested by PCR. Successfully transfected cells gave an amplifiedproduct of 167-bp as shown with positive controls pHaMDR1 (FIG. 8, upperrow, lane 2), NIH3T3 colchicine resistant cells (FIG. 8, upper row, lane3), and NIH3T3 cells transfected with the human MDR1 cDNA using thecalcium-phosphate precipitation method (FIG. 8, upper row, lane 4). Apositive band was obtained with in vitro DLS/MDR transfected BMC thatwere selected for 48 hours in 30 ng/ml vincristine (FIG. 8, upper row,lane 7). FIG. 8 shows representative positive samples that were obtainedfrom mouse hematopoietic tissues. Positive bands were detected in BMC,spleen, and PB cells (FIG. 8, upper row, lanes 10, 11, 12 respectively)of an DLS/MDR in vivo treated mouse, 15 days after i.v. injection; aswell as in BMC and spleen cells (FIG. 8, upper row, lanes 13, 14respectively) of a reconstituted mouse, 15 days post-BMT with DLS/MDR1transfected BMC. The MDR1 gene was still detected in BMC 30 dayspost-transplantation in the one mouse tested (FIG. 8, lower row, lane2). None of the control animals DLS/Neo i.v. transfected (FIG. 8, lowerrow, lane 5, 6, 7; BMC, spleen cells, and PB cells respectively), ortransplanted with DLS/Neo transfected BMC (FIG. 8, lower row, lanes 8,9: BMC, and spleen cells respectively) showed any positive band 15 dayspost-transfection nor 30 days post-BMT (FIG. 8, lower row, lane 3: BMC).

In DLS/MDR in vivo treated mice, specific band for the human MDR1 wasdetected in 5 of 6 BMC samples, 4 of 5 spleen cells, and 4 of 7 PB cellstested. BMC from one in vivo treated mouse gave a positive band whenanalyzed 28 days post-i.v. injection with DLS/MDR. In the group oftransplanted mice, human MDR1 specific band was detected in 3 of 4 BMC,2 of 3 spleen cells, and 2 of 2 PB tested cells. All in vitrotransfected BMC turned out positive for human MDR1 whether before orafter being drug selected (4 of 4 and 2 of 2 respectively).

EXAMPLE 11 In vitro DLS/MDR Transfection Leads to Expression of P-gp inBMC

Transfection of mouse BMC with DLS/cDNA for 4-5 days did not affect themorphology nor the number of cells collected when compared with theuntransfected BMC control. However, when the cells were subjected toselection with 30 ng/ml of vincristine for 48 hours, the numbers ofviable cells obtained varied greatly. From a total of 3×10⁶ cells platedbefore the selection pressure was added, only 1.5×10⁴ cells, and 2×10⁴viable cells were counted in the untransfected, and in the DLS/Neotransfected control cells respectively. In contrast, 2×10⁶ cellsremained viable in the DLS/MDR transfected BMC. Using the human P-gpspecific monoclonal antibody MRK16, and G2CL monoclonal antibody as anirrelevant negative control antibody, FACSort analysis was performed ofDLS/MDR transfected BMC not subjected (FIG. 9A and subjected toselection with 30 ng/ml of vincristine for 48 hours (FIG. 9B). Thehistogram in FIG. 9A shows that after transfection, MRK16 monoclonalantibody stained 15% of the cells positively above the backgroundfluorescence level. Moreover, the whole histogram representing BMCstained with MRK16 monoclonal antibody appeared positively displacedwhen compared to the histogram representing the cells stained with G2CLmonoclonal antibody. Positive staining was noted each time the analyzedwas done (5 of 5).

Using FACSort analysis, we demonstrated the function of the gene productin a rho123 efflux assay as early as 5 days post-transfection withDLS/MDR. As shown in FIG. 10, 3 hours after exposure to rho123 thefluorescence level of the non-selected in vitro DLS/MDR transfectedcells was lower than that of the control BMC transfected with DLS/Neo.

Staining for P-gp expression

Collected cells were resuspended, washed in PBS (Gibco Laboratories)supplemented with 0.1% bovine albumin (Sigma) and incubated with MRK16(generously provided by Hoecht-Japan), a mouse IgG₂ monoclonal antibodyspecific for an external epitope of the P-gp. As a negative control,G2CL (Becton Dickinson, Calif.), a mouse IgG_(2a) monoclonal antibodywas used. Non-specific binding to mouse cells were prevented by the useof 24G2, a rat anti-mouse Fc receptor monoclonal antibody (Pharmingen,San Diego, Calif.) that was incubated for 10 minutes prior to theaddition of the MRK16 and G2CL. After a 30 minute incubation at 4° C.,the cells were washed and incubated with a secondary goat anti-mouse IgGfluoro-iso-thiocyanate conjugated (GαM IgG-FITC) antibody for another 30minutes at 4° C. After a final wash, the cells were analyzed using aFACSort (Becton Dickinson, Calif.), and fluorescence intensity levelswere illustrated as histograms plotted against the X axis on alogarithmic scale, with the relative cell number displayed along the Yaxis.

Colony forming unit assay in methylcellulose

An assay for hematopoietic progenitor cells able to form colonies(CFU-Mix) was performed using an established method (Wong et al. 1986PNAS 83;3851-4). 1×10⁴ BMC were plated in semisolid medium containing0.9% methylcellulose (StemCell Technologies Inc., Vancouver, Canada),10% calf serum, 1% serum, 1% glutamine, 1% penicillin, 1% streptomycin,100 ng/ml mouse erythropoietin factor (Sigma), 100 ng/ml mouse II3, and100 ng/ml mouse G-CSF (Pharmingen, San Diego, Calif.). In eachexperiment, all cells were plated in triplicate wells of a 96-wellmicrotiter plate (Nunc). CFU-Mix that were clearly expended wereenumerated using an inverted microscope after incubation at 37° C. in ahumidified 5% CO₂ atmosphere for 10 to 12 days.

In vitro transfected BMC, were analyzed for their ability to formcolonies in semisolid medium in the presence or absence of vincristine.Although conditions varied somewhat (these clonogenic assays were donewith cells from three different in vitro transfection experiments), whenno selection was applied there were typically between 10 to 18 CFU-Mixper 10⁴ total BMC plated. As in vitro controls, non-transfected orDLS/Neo transfected BMC were assayed. FIG. 11, panel A, representtypical colonies obtained from DLS/Neo transfected BMC. Their number andmorphology was comparable to the colonies formed from untransfected andDLS/MDR transfected BMC (FIG. 11, panel B). When BMC clonogenicpotential was tested under selective pressure of 20 ng/ml ofvincristine, no colonies formed from the untransfected, nor from theDLS/MDR transfected BMC (FIG. 11, panel D). Table 1, panel A, showsabsolute numbers of CFU-Mix obtained from 1×10⁴ plated BMC.

                  TABLE 1                                                         ______________________________________                                               normal BMC                MDR1                                                         day 5 in culture                                                                   Neo BMC day 5                                                                                        BMC day 5                         A               not transfected                                                                    post transfection                                                                                  post transfection                   ______________________________________                                        no           17.8 ± 8.9                                                                                 13.2 ± 8.3                                                                             12.1 ± 4.2                        selection                             p-0.06                                  10 ng/ml                                                                                       13.4 ± 8.13                                                                           7.7 ± 7.1                                                                               9.8 ± 3.9                         vincristine                                                                                                                   p = 0.3                       20 ng/ml                                                                                         0                                                                                                               3.6 ±  2.6            vincristine                                                                                   0%                               p = 0.007                                                     (29.7%)                                      ______________________________________                                                                               MDR1                                                            Neo BMC day 5                                                                                      BMC day 5 post                                        post transfection, 48                                                                         transfection, 48                        B                      hours preselection                                                                              hours preselection                   ______________________________________                                        no           9.6 ± 9.9                                                                                   5.4 ±  2.1                                                                       39.7 ± 9.2                             selection                                                                                                                    p = 0.007                      10 ng/ml                                                                             3.8 ± 6.3 2.6 ± 1.3 33 ± 4.0                                  vincristine                      p = 0.000                                    20 ng/ml                                                                             0.6 ± 1.1 0.7 ± 0.5 26.5 ± 7.4                                vincristine                                                                          6.25%        12.9%        P = 0.000                                                                     (66.7%)                                      ______________________________________                                    

Under no selection, the number of colonies obtained from theuntransfected, DLS/Neo transfected, and DLS/MDR transfected BMC weresimilar, with 17.8+/-8.8, 13.2+/-8.3, and 12.1+/-4.2 CFU-Mixrespectively. When 10 ng/ml of vincristine was added to themethylcellulose, only 13.4+/-8.13 CFU-Mix were counted from theuntransfected, and 7.7+/-7.1 CFU-Mix were counted from the DLS/Neotransfected BMC. In contrast, 9.8+/-3.9 (p=0.3) CFU-Mix grew from theDLS/MDR transfected BMC. Most significant was the difference observedwhen the BMC were tested under 20 ng/ml of vincristine. No colonies grewfrom the untransfected, nor from the DLS/Neo transfected BMC, whereas3.6+/-2.8 (p=0.007) of the DLS/MDR transfected BMC were able to formCFU-Mix. This indicated a efficiency of transfected of 29.70%.

After transfecting the cells in vitro, the cells were subjected to 30ng/ml vincristine for 48 hours. This significantly enriched for thepopulation of MDR1 positive progenitor cells, as shown in Table 1, panelB. Under no selection, 39.7+/-9.2 (p-0.007) CFU-Mix were counted fromthe DLS/MDR transfected BMC. Whereas, only 9.6+/-9.9 and 5.4+/-2.1colonies grew from the untransfected BMC, the number of CFU-Mix droppedslightly to 33+/-4.0 (p=0.000) and 26.5+/-7.4 (p=0.000) when 10 ng/ml or20 ng/ml of vincristine was added respectively. This shows that 66.7%positive cells were selected by pre-selecting the transfected cells. Incontrast, at 10 ng/ml vincristine the numbers of CFU-Mix in theuntransfected and DLS/Neo transfected BMC dropped to 3.8+/-6.3 and2.6+/-1.3 respectively, and at 20 ng/ml vincristine the numbers droppedto 0.6+/-1.1 and 0.3+/-0.5 respectively.

EXAMPLE 12

Transplantation of in vitro DLS/MDR transfected BMC and systemicdelivery of DLS/MDR is followed by P-gp expression in mouse BMC

Analysis of BMC obtained 5 days post-BMT from recipients of DLS/MDRtransfected BMC showed no positively stained cells with MRK16, and GαMIgG-FITC antibody. However, 15 days post-BMT as shown on FIG. 12A,staining of DLS/MDR BMC from a transplanted mouse demonstrated higherlevels of fluorescence than that of BMC taken from a mouse transplantedwith DLS/Neo transfected cells, with 19.1% of the cells staining abovethe control levels. P-gp on BMC was still detectable by FACSort analysis25 days post-BMT (FIG. 12B), with 21% of the cells staining positivelyin that mouse BMC.

FIGS. 12C, and 12D represent data obtained by FACSort analysis of BMCfrom two DLS/MDR injected mice stained with MRK16, and GαM IgG-FITC 12days (8 positive out of 9 tested), and 25 days (2 positive out of 2tested) post-i.v. injection respectively. At both time points, BMC fromDLS/MDR transfected mice demonstrated positive specific staining forP-gp when compared to the BMC obtained from DLS/Neo transfected mice. Atday 12, DLS/MDR mouse cells stained positively (21.3i) compared to theDLS/Neo transfected mice. 25 days post-injection, 14.2% of the BMCstained positively.

EXAMPLE 13

P-gp expression in different BMC lineages after BMT and systemicdelivery

15 days post-BMT, BMC were harvested from three mice, and analyzed forP-gp expression in different cell lineages. BMC were stained with MRK16and GαM IgG-FITC. FIG. 13, top panel, shows a dot plot fluorescencerepresenting cell size (FSC) plotted against cell density (SSC) thatallowed us to differentiate three morphologically different populationof BMC. Same cell distribution were seen in the DLS/Neo FIG. 13, (left),and the DLS/MDR FIG. 13, (right) transfected BMC: the lymphocytes shownas small sized cells of low density (R1), the monocyte as large sizedcells of intermediate density (R2), and the granulocyte as intermediatesized cells of high density (R3). By gating each sub-population, therespective MRK16 fluorescence was observed (FIG. 13, lower panel). TheFACSort data revealed that in each sub-population, namely thelymphocytes, the monocyte, and the granulocyte, most of the cellsexpressed P-gp.

Similar results as in the transplanted mice were found after analysis ofBMC obtained from in vivo transfected mice 12 days post-i.v. injection(FIG. 14). When populations of BMC were transfected, all threehematopoietic cell populations gated, namely the lymphocytes (R1), themonocyte (R2), and the granulocyte (R3) specifically stained with MRK 16when compared to the background DLS/NEO injected control BMC.

BMT with DLS/MDR transfected BMC and in vivo treatment with DLS/MDRleads to P-gp expression in hematopoietic progenitor cells

At several time points after reconstitution the presence of drugresistant clonogenic progenitor hematopoietic cells were tested (two orthree bone marrow transplanted mice from each group were tested at eachtime point). The results are presented in Table 2, panel A, representingthe means values +/- standard deviation of colonies obtained with 20ng/ml vincristine.

                                      TABLE 2                                     __________________________________________________________________________    4 days      10 days                                                                              15 days   31 days                                          post-BMT      post-BMT                                                                            post-BMT  post-BMT                                        A    Neo                                                                              MDR Neo                                                                              MDR Neo MDR   Neo                                                                              MDR                                           __________________________________________________________________________    20 ng/ml                                                                           0     0                                                                                         5.8 ±+-.                                                                         0  2.5 ±                                      vincristine                                                                                                2.4                                                                               0.7                                                                   p = 0.0000                                                                           p = 0.1                                       4 days      10 days                                                                              21 days   36 days                                          post-injection                                                                            post-injection                                                                         post-injection                                                                         post-injection                                  B    Neo                                                                              MDR Neo                                                                              MDR Neo MDR   Neo                                                                              MDR                                           __________________________________________________________________________    20 ng/ml                                                                           0  0   0  13.9 ±                                                                         0   7.7 ±                                                                            0  2.4 ±                                      vincristine    3.3     5.0      2.1                                                          p = 0.1 p = 0.1  p = 0.04                                      __________________________________________________________________________

With no drug selection added, BMC from DLS/Neo, and DLS/MDR BMT micecontained similar numbers of CFU-Mix (i.e.: at day 15 post-BMT, Neo:22.7+/10.4, and MDR: 27.3+/-3.5). At 20 ng/ml vincristine, no coloniesgrew on day 4, or day 10 post-BMT from any DLS/Neo, or DLS/MDR recipientmice. Still, the results indicate that the transfection efficiency of8.80 is real.

Injected mouse BMC, was also analyzed for its ability to form coloniesin semisolid medium in the presence or absence of vincristine atdifferent time points post-injection. As controls, DLS/Neo injected micewere assayed. With no drug selection added, BMC from DLS/Neo, andDLS/MDR injected mice contained similar numbers of CFU-Mix (i.e.: at day21 post-DLS/MDR and DLS/Neo injection, 47.8+/-21.5, and 59.4+/-24.3CFU-Mix were counted respectively; at day 36 post-injection, 30+/-12.1,and 28.2+/-6.3 colonies were counted in the DLS/MDR, and DLS/Neoinjected mice had no CFU-Mix at days 4, 10, 21 and 36 post-treatmentrespectively. In contrast, the DLS/MDR treated mice, that grew nocolonies at day 4 post-injection, had 13.9+/-3.3, 7.7+/-5, 2.4+/-2.1CFU-Mix, at day 10, 21, and 36 post-injection respectively. Thedifference was significant (p, <0.05) only for the value taken at day36, indicated an actual transfection efficiency of 8%.

EXAMPLE 14

In vivo administration of DLS-liposomes into Balb-C mice.

Plasmid DNA. Firefly "Photinus pyralis" peroxisomal luciferase gene(luc) was used as a report gene for the monitoring of transgeneexpression levels. In preliminary studies episomal vectors wereconstructed containing a genomic fragment of the human papovavirus, BKV.This fragment included the BKV viral early region and origin ofreplication, the large T antigen, and later viral capsid proteins.Sequence encoding the firefly luciferase (luc; 1.7 kb Bam HI-Sal Ifragment of PGEM-luc [Promega]) gene was inserted under the control ofthe Rous sarcoma virus (RSV) promoter and the polyadenylation signal andtranscriptional termination sequences from SV40. The resultantepisomal/reporter expression vector was termed pBKd1RSv-luc. Forsubsequent studies, a modified BKV plasmid containing the luc gene wasconstructed in which the entire sequences coding for the late BKV capsidproteins (VP1, VP2 and VP3) (Seif, I. et al. (1979) Cell 18, 963-977)were deleted to remove the expression of these potentially immunogenicproteins. The resultant episomal/reporter expression vector was termedpBKd2RSV-luc. In addition, a second series of pBKd1 and pBKd2 vectorswere made in which luc expression was placed under the control of theenhancer/promoter sequences from the immediate early gene of the humancytomegalovirus (CMV) and the polyadenylation signal and transcriptionaltermination sequences from the bovine growth hormone gene. The resultantepisomal/reporter expression vectors were termed pBKd1CMV-luc andpBKd2CMV-luc, respectively. The non-episomal pRSV-luc containing lucunder the control of the RSV long terminal repeat was constructed aspreviously reported (DeWett, J. et al. (1987) Mol. Cell. Biol. 7,725-737). pCMVintlux plasmid encodes the luc under the control of humanCMV immediate-early promoter with intron A (Manthorpe, M. et al (1993)Hum. Gene Ther. 4, 419-431).

Preparation of the DLS Liposomes. Liposomes were formed by mixing 1 mgdioctadecyl-amidoglycyl spermidine and 1 mg dioleoyl-phosphatidylethanolamine. After thorough stirring, the mixture was evaporated todryness in a round-bottomed borosilicate tube using a rotary vortexevaporator under vacuum. Then the dry lipid film was hydrated with amaximum volume (60 μl/mg lipid) of a solution containing 160 μg plasmidDNA and was slightly vortexed. After incubation at room temperature for15 min, the resulting suspension was vigorously mixed by vortex.Subsequent liposomal DNA preparation was then diluted in 150 mM NaCl,and kept at 4° C. Liposomes appeared to range from 200 to 3,000 nm indiameter as determined by transmission electronic microscopy. DLSliposomes consist of multilamellar bilayers vesicles which may complexas well as encapsulate DNA. The entrapment rate was found to be 88±8%(mean ±S.D.) of the initial DNA input dose. This type of liposomes wereused in examples 14-17.

In Vivo Gene Transfer. Plasmid-DNA in DLS-liposomes-2 liposome wasadministered by a single injection of 100 to 600 μl total volume in thetail vein of 4-6 week old female Balb/C mice. Control mice received 150mM NaCl solution. Mouse tissues were collected in 2 ml Eppendorf tubes,quickly frozen on dry ice and stored at -70° C. until examined.

Plasmid vector containing the luciferase gene as a marker gene weredelivered by DLS-liposomes in Balb-C mice. Various formulations ofliposomes encapsulated plasmid at various DNA/lipid ratios were assayed.In these experiments transgene expression has been assayed in liver,lung and spleen. Luciferase activity was determined by bioluminescencemeasurement (2-3 mice/point). More than 100 mice have been studied. PCRanalysis showed the long lasting expression of the lucriferase gene inall tissues tested (lung, liver, heart, spleen, skeletal muscle, bloodcells, bone marrow, and ovary) up to at least 2 months post-injection.Only episomal replicating DNA vectors showed positive results.

EXAMPLE 15

Intravenous administration of DLS-liposomes containing plasmid DNA: DoseResponse Study

DNA Dose-Response. To assess for treatment-related toxicity and todetermine the optimal liposomal DNA amount to be injected, dose-responseexperiments were carried out. Episomally replicative pBKd1RSV-lucplasmid DNA encapsulated in DLS liposomes was administered i.v. intomice. Luciferase activity was determined 4 days post-injection in liver,lung, and spleen (FIG. 15). Luciferase activity could be detected inthese tissues even when the amount of injected DNA amount was as low as25 μg/mouse. No significant difference in luc gene expression betweentissues was observed at this concentration while at 100 μg DNAinjected/mouse a significant difference in activity was observed betweenlung, liver and spleen (1700±100, 1020±300 and 490±130 fg luciferase/mgprotein, respectively; mean ±S.D.). In contrast to lung and spleen wherethe transgene expression reached a plateau at approximately 200 μg and50 μg DNA, respectively, there was a decrease in transgene expression inthe liver with DNA deliveries greater than 100 μg, perhaps due totoxicity of the liposomal DNA in liver cells or a greater accumulationof liposomal DNA in this organ. No luciferase activity could be detectedin liver or lung tissue of mice injected with 100 μg "naked"pBKd1RSV-luc plasmid or the pBKd1RSV plasmid construct containing thelacz gene instead of luc presented in DLS liposomes. Macroscopicallyslight toxicity was observed (enlarged spleen and pale liver) when100-500 μg of liposomal DNA were injected into mice. Marked toxicity(grayish lung, tissue damage in heart and occasional death) was observedwhen more than 150 μg liposomal DNA were administered. No gross ormicroscopic anatomical pathology was observed at liposomal DNA doses<100 μg. The DNA/lipid ratio was critical as an increase in positivelycharged lipids may contribute to serious toxicity. The efficiency of DLSliposomes to deliver the transgene depended directly upon the cationiclipid amount in the preparation. Transgene expression in lung greatlyincreased from 55±20 to 3750±950 fg luciferase/mg protein (mean ±S.D.)when the DNA/lipid ration (w/w) decreased from 0.32 to 0.08. A ratio of0.08 was optimal and was used throughout this work.

EXAMPLE 16

Effect of the Route of Administration. pBKd1RSV-luc plasmid DNA (25 μg)were injected intravenously (i.v.) (tail vein), Intraperitoneally (i.p.)and subcutaneously (s.c.) and luciferase activity was measured 4 dayspost-injection. No significant difference was observed in the spleen byuse of different routes of administration but slightly more activity wasdetected in the liver and lung with the i.v. route (FIG. 16). S.c.injection appeared to be the less efficient route of administration.I.p. administration resulted in a preferential targeting to the spleen.

Immunohistochemical Detection of Luciferase Expression. Luciferaseexpression was detected in tissue samples from mice treated with 75 μgpBKd2CMV-luc and sacrificed 6 days post-injection by immunohistochemicalstaining using a polyclonal antibody against luciferase protein (FIG.17). The recombinant protein was detected in all tissues tested althoughthe percentage of positive cells varied: heart, >75%; spleen andliver, >50%; and colon and lung >10%. The pattern of transgeneexpression was generalized throughout the heart (FIG. 17A, vascularendothelial cells and myocytes), the liver (FIG. 17B, hepatocytes,Kupfer cells and endothelial cells) and the spleen. Although stainingintensity was lower in the colon (FIG. 17D) and in lung, staining wasalso diffuse in these tissues. In separate experiments, 50 pgpBKd1RSV-luc in DLS liposomes were injected i.v. in immunodeficient micebearing Kaposi's sarcoma (KS Y-1) tumor cells (Lunardi-Iskandar, Y. etal. (1995) Nature 375, 64-68). The luciferase protein was detected intumor by immunostaining with more than 10% of the tumor cells beingpositive (FIG. 17E). No positive cells were found in liver (FIG. 17C) orKS Y-1 tumor cells (FIG. 17F) when luciferase antibody was replaced bynormal rabbit serum as a control.

EXAMPLE 17

Stability of Luciferase Expression in Different Tissues. pBKd2CMV-lucplasmid DNA (75 μg) was administered i.v. and luciferase activity wasmeasured in liver, lung, spleen and heart at different times (FIG. 18).

                                      TABLE 3                                     __________________________________________________________________________    Detection of the luciferase gene in mouse tissues by PCR analyses             PCR  Liver                                                                             Lung                                                                             Spleen                                                                            Heart                                                                            Muscle                                                                            BMC                                                                              PB  Brain                                                                            Ovary                                        __________________________________________________________________________    6 days                                                                             2/2 2/2                                                                              2/2 2/2                                                                              3/3 3/3                                                                              3/3 ND 3/3                                          2 weeks                                                                               ND                                                                                ND                                                                                ND                                                                               ND                                                                                2/2                                                                              2/2                                                                            ND   2/2                                                                               2/2                                       1 month                                                                               2/2                                                                              1/2                                                                               1/2                                                                              1/2                                                                               2/2                                                                               1/2                                                                            1/2                                                                               2/2                                                                                1/2                                       2 months                                                                             2/2                                                                               2/2                                                                               1/2                                                                              1/2                                                                               2/2                                                                               2/2                                                                            1/2                                                                               0/2                                                                                0/2                                       3 months                                                                             1/1                                                                               1/1                                                                               1/1                                                                              1/1                                                                               0/1                                                                               0/1                                                                            0/1                                                                               0/1                                                                                0/2                                       6 months                                                                             0/2                                                                               0/2                                                                               0/2                                                                              0/2                                                                               0/2                                                                               0/2                                                                            0/2                                                                               0/2                                                                                ND                                        __________________________________________________________________________     Number of positive tissues/Number of treated mice.                            75 μg of DLSpBKd2CMV were administered i.v. in mice.                       BMC, bone marrow cells; PB, peripheral blood; ND, not determined.        

Transgene expression was maximal between 6 and 15 days post-injection inlung, spleen and heart and then gradually declined over 3 months.Luciferase activity was low and constant for up to 3 months in theliver. The luciferase activity was also detected at 6 days in otherorgans such as the skeleton muscle, brain, bone marrow cells (BMC) andperipheral blood (PB) (398±130, 192±103, 2220±73 and 160±46 fg/mgprotein, respectively; mean ±S.D.). Peripheral blood mononuclear cellsobtained from whole blood after ficoll purification contained 900 fg/mgprotein which corresponded to approximately 1000 detected molecules ofluciferase/cell.

PCR analysis confirmed the presence of luciferase activity since alltested tissues were positive for luc 6 days post-injection (Table 3).The transgene was found for up to 1 month in brain and ovary, for up to2 months in muscle, BMC and PB, and for up to 3 months in lung, liver,spleen and heart. In addition, kidney and colon tissues were positive 2months post-injection.

Luc expression was detected by RT-PCR analysis. As shown in FIG. 19,liver, spleen lung were positive 3 months post-injection.

Effect of Plasmid Construct and Promoter on Transgene Expression. Theexpression of six luciferase plasmids in different organs were comparedbeginning 6 days after a single i.v. injection of 75 μg DNA deliveredwith DLS liposomes. The results at 6 days are summarized in Table 4 andshow that when using the pRSV-luc plasmid luciferase activity was onlydetected in lung. In contrast, luciferase activity was high in alltissues tested (liver, lung, spleen and heart) when pCMVintlux was used.The four BKV-derived episomal plasmids showed slightly less orequivalent reporter gene activity. The CMV promoter yieldedsignificantly higher levels of gene expression in spleen and lowerlevels in lung when compared with the RSV promoter driving the sameplasmid DNA construct.

                  TABLE 4                                                         ______________________________________                                        Plasmid construct and promoter effects on transgene expression                           Liver  Lung     Spleen   Heart                                     ______________________________________                                        pRSV-luc     0        ++       0      0                                       pCMVintlux        ++        +++                                                                                   ++                                                                               +++                                    pBKd1RSV-luc     +                     +++ ++                                 pBKd1CMV-luc     ++    +*      +++.sup.†                                                                     +++                                     pBKd2RSV-luc     ++    +++      +      ++                                     pBKd2CMV-luc    +      ++.sup.‡                                                                   +++.sup.§                                                                                  ++                           ______________________________________                                         Relative luciferase activity: 0, not detectable; +, 0-0.1 pg/mg protein;      ++, 0.1-1.0 pg/mg protein; +++, higher than 1.0 pg/mg protein. i.v.           administration, activity determined 6 days postinjection. pBKd1CMVluc         versus pBKd1RSVluc in *lung (p = 0.011) and in .sup.† spleen (p =      0.001), and pBKd2CMVluc versus pBKd2RSVluc in .sup.555 lung (p = 0.187)       and in .sup.517 spleen (p = 0.021).                                      

The time course of luc expression in mouse tissues from thenon-replicative pCMVintlux and episomal pBKd2CMV-luc plasmid constructswas followed after a single i.v. injection of 75 Ag DNA (FIG. 17). Lucproduct expressed from pBKd2CMV-luc was detected in lung, liver, spleenand heart for up to 2-3 months post-injection. No detection ofluciferase activity was observed in these tissues 1 month afterinjection of pCMVintlux.

EXAMPLE 18

Intraperitoneal administration of DLS-liposomes containing DNA.

Luciferase gene expression was detected in spleen 3 days post-injection.The dose injected per mouse was 100 ug. No toxicity was detected.

EXAMPLE 19

Inhibition of KS Y-1 cell tumorigenicity.

The present invention can be used in the therapy of Kaposi's Sarcoma("KS"). Two KS cell lines, showing tumorigenic properties in vitro andin vivo, have recently been established. One cell line, KS Y-1, wasderived from a lesion of an HIV-infected individual. The second cellline, KS N1506, was derived from a lesion of a non-HIV associatedimmunodepressed individual. High amounts of IL-6, IL-8, and VEGF areproduced in these cell lines. Correspondingly, high levels of thesecytokines have also been found in the serum of AIDS-KS patients. In thisexample, antisense oligo(dN) was used as a specific molecular tool toinhibit KS cell production of these factors.

0.1 uM VEGF antisense phosphodiester oligodeoxynucleotides encapsulatedin DLS-liposomes completely blocked KS Y-1 cell colony formation insemi-solid culture. Lipofectin™liposomes required 7-10 fold higherconcentration to achieve the same inhibitory effect.

What is claimed is:
 1. A composition comprising a bi- or multi-layermembrane surrounding an internal aqueous liposome comprising at leastone cationic lipopolyamine and at least one neutral lipid provided in amolar ratio range said ratio from about 0.02:1 to about 2.0:1.
 2. Acomposition according to claim 1 wherein the lipopolyamine comprises aquaternary or tertiary polyamine lipid.
 3. A composition according toclaim 2 wherein at least one cationic lipopolyamine is selected from thegroup consisting of 2,3-dioleyloxy-N[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoracetate, N[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethyl-ammoniumchloride and Spermine-5-carboxy-glycinediotadecylamide.
 4. A compositionaccording to claim 3 wherein the cationic lipopolyamine comprises atleast one spermine-5-carboxy-glycinedioctadecylamide.
 5. A compositionaccording to claim 1 wherein the neutral lipid is a neutral aminophospholipid.
 6. A composition according to claim 5 wherein the neutrallipid is selected from the group consisting of dioleylphosphatidylethanolamine and phosphatidylethanolamine.
 7. A composition of claim 1wherein the cationic lipopolyamine comprisesspermine-5-carboxy-glycinediocadecylamide and the neutral lipidcomprises dioleylphosphatidyl ethanolamine.
 8. A composition of claim 1wherein the cationic lipopolyamine comprisesspermine-5-carboxy-glycindioctadecylamide and the neutral lipidcomprises phosphatidylethanolamine.
 9. A composition according to claim1 or 7 further comprising a biologically active agent.
 10. A compositionaccording to claim 9 wherein the biologically active agent is selectedfrom the group consisting of a therapeutic agent, a protein or a nucleicacid.
 11. A composition according to claim 10 wherein the nucleic acidis selected from the group consisting of a chromosome of a chromosomalfragment, a deoxyribonucleic acid, a ribonucleic acid, a ribozyme, anoligonucleotide, an anti-sense oligonucleotide, a plasmid DNA or anucleic acid viral in origin.
 12. A method of preparing a liposomecomprising the steps of:(a) mixing a cationic lipopolyamine with aneutral lipid in a molar ratio range of about 0.02:1 to about 2.0:1,forming a mixture; (b) evaporating the mixture to dryness, forming adried film; (c) adding a biologically active agent; (d) rehydrating thedried film with said biologically active agent forming the liposome. 13.The method of claim 12 wherein the biologically active agent is anucleic acid solution provided in a ratio of 40-240 microgram nucleicacid per milligram lipid.
 14. The method of claim 13 wherein saidnucleic acid is provided in a concentration of about 1-3 mg/ml.
 15. Themethod of claim 13 further comprising rehydrating the dried film in asolution having a pH of 5.5-6.5.
 16. The method of claim 13 wherein theaqueous solution is water.
 17. A method of introducing a biologicallyactive agent into cells of a subject comprising administrating to thesubject an effective amount of a composition according to claim
 9. 18.The method of claim 17 wherein the biologically active agent is anucleic acid.
 19. The method of claim 18 wherein the nucleic acid is aDNA.
 20. A composition of claim 9 wherein the biologically active agentcomprises adenovirus particles.
 21. A composition according to claim 9further comprising adenovirus particles.